Vertebrate animals can suppress expression of CpG dinucleotide in their genomic DNA sequence or have a cytosine residue of the expressed CpG dinucleotide methylated (Krieg, Ann. Rev. Immunol., 2002, 20, 709; McClelland & Ivarie, Nucleic Acids Res. 1982, 23, 78). Meanwhile, a microbial CpG dinucleotide is expressed at a normal rate and not methylated, and therefore it enables to detect microbial infection in vertebrates using the difference in the levels of the expressed CpG dinucleotide between vertebrate animals and microorganisms.
A microbial genomic DNA is recognized by dendritic cells or B cells expressing the TLR9 via toll-like receptor 9 (TLR9), a pattern recognition receptor, and eventually activates an innate immune system of a host cell. The innate immune system is endowed with a mechanism for removing cancer cells as well as a self defense mechanism against microbial or parasitic infections, and thus it is expected to develop a carcinostatic immunological adjuvant capable of inducing anti-cancer activity of an immune system by properly modifying the CpG ODN.
Studies for activating immune system using the CpG ODN have been actively progressed for the past few decades. The CpG ODN contains at least four bases at both 5′- and 3′-termini with reference to the CpG dinucleotide as the center, and immunoactivity of the CpG ODN is characterized by the base sequence. The CpG ODN is subdivided into two groups, K type and D type. K type ODN stimulates myeloid lineage cells and B cells thus resulting in their proliferation or secretion of immunoglobulin M or IL-6 (Klinman et al., Microbes Infect., 2002, 897-901). On the other hand, D type ODN activates monocytes to be differentiated into dendritic cells or stimulates natural killer cells to secrete IL-6 (Klinman et al., Eur. J. Immunol., 2002, 32, 2617-22; Gursel et al., J. Leukoc. Biol., 71, 813-20, 2002). Further, the D type ODN activates B220+ dendritic cells to produce IFN-α while TLR9+ B220+ dendritic cells release IL-12 in response to D type ODN. These results suggest that there might be several pathways to improve immune activity of the CpG ODN by stimulating specific immunocytes (Hemmi et al., J. Immunol., 2003, 170, 3059-3064; Kerkman et al., J. Immunol., 2003, 170, 4465-4474).
Various types of CpG ODNs have been designed to redirect the pathologic condition such as infection, autoimmune disease and cancer. Of these, the strategy toward the development of immunotherapeutic CpG ODN against cancer can rely upon effector cells mainly stimulated by CpG ODN. To augment cell-mediated immunity using CpG ODN, the following two methods are commonly exploited.
The first method is to augment a local or systemic immunity via an activation of naive or professional dendritic cells. Cancer cells down-regulate their antigen presentation capabilities to escape an immune surveillance system of a host cell, thereby enabling to survive in the host cell taking advantage of the fact that host cytotoxic T cells are unable to recognize them. To solve this problem, there has been developed a method that immunizes a host with a strong immunogenic peptide in a cancer antigen together with the CpG ODN as an immune adjuvant. By this method, the dendritic cells with high antigen presentation activity can uptake the peptide of cancer antigen and when activated by the CpG ODN, leading to activating cytotoxic T cells. The activated cytotoxic T cells can then effectively eliminate the cancer cells. It has been reported that this method can kill RMA from a mouse (Stern et al., J. Immunol., 2002, 168, 6099-6105). IFN-γ plays an important role in these immune responses. Although this is not done through activation of dendritic cells, it still enables to activate anti-cancer immunity same as in the mechanism of dendritic cells by rendering CpG ODN 2006 to directly work on B cells as well as to increase the expression of a costimulator which can induce an interaction between B and T cells (Jahrsdorfer et al., J. Leukoc. Biol., 2001, 69, 81-88).
The other method is to augment innate immunity via activation of natural killer cells. It is possible to activate cytotoxic T cells by activating dendritic cells by introducing a cancer antigen from the outside, but it is essential to present the cancer antigen on the surface of MHC class I molecule for eliciting cytotoxicity from cytotoxic T cells. However, in many cases the level of presenting caner antigens on the surface of cancer cells is too low to elicit cytotoxicity from cytotoxic T cells, and therefore the method for removing cancer cells by activating the dendritic cells often becomes ineffective. To overcome this limitation, it has been suggested to activate natural killer cells which exert cytotoxicity regardless of cancer antigen presentation on the surface of a cancer cell. Further, the activated natural killer cells activate monocytes or macrophages, leading to activation of antigen-independent anti-cancer immune system. It has been reported that CpG ODN 1584 administration in vivo blocks the metastasis of NK sensitive B16.F1 melanoma whereas CpG ODN 1826 injection effectively rejects NK resistant EL4 lymphoma in an in vivo_mouse tumor model using the above method (Ballas et al., J. Immunol., 2001, 167, 4878-4886). Further, when ODN 1826 was directly injected into a tumorigenic lesion after induction of C26 colon carcinoma mass in BALB/C mice, the size of the tumor was markedly decreased. This data shows that peritumoral CpG ODN monotherapy elicits a strong CD8 T cell response and innate effector mechanisms that seem to act in concert to overcome unresponsiveness of the immune system toward a growing tumor. (Heckelsmiller et al., J. Immunol., 2002, 169, 3892-3899). In contrast, it is difficult to eliminate the murine 38C13 B cell lymphoma in vivo by means of CpG ODN monotherapy. However, if a monoclonal antibody specific to 38C13 lymphoma antigen is treated with the CpG ODN, the activated natural killer cell is capable of efficiently removing 38C13 lymphoma by exerting antibody-dependent cell cytotoxicity (Woodridge et al., Blood, 1997, 89, 2994-2998).
Meanwhile, humoral immunity against cancer can be induced by using an antigen or an equivalent thereof together with the CpG ODN as an immunostimulant. Trastuzumab and rituximab have been known as commercial monoclonal antibodies specific to HER-2 protein over-expressing cancer cell and non-Hodgkins B cell lymphoma, respectively. Recently, the combination therapy with the CpG ODN plus tumor antigen specific antibody demonstrated the potent tumor rejection preclinically and is now entering into clinical trials. (Jahrsdorfer et al., Sem. Oncol., 2003, 30, 476-482).
The CpG ODN has strong innate immunoactivity due to the nucleotide sequences containing CpG dinucleotides present at 5′- and 3′-termini. However, since the CpG ODN itself is of a wild-type structure having no difference from a structure of in vivo DNA molecule, it does not show any cytotoxicity. However, there is a disadvantage in the CpG ODN that it is easily degraded in vivo due to its wild-type structure, consequently leading to reducing half-life of immunoactivity. Although it is possible to increase a daily dosage of CpG ODN to overcome this drawback, it is not economical. It has been reported that when a phosphodiester bond known as a backbone of DNA molecule is changed into a phosphorothioate bond during the synthesis of CpG ODN, immunoactivity of the CpG ODN is amplified about 10 to 100-folds (Sester et al., J. Immunol., 2000, 165, 4165-4173). However, this method has the problems of bringing about cytotoxic effects to immune cells as well as changes in immunoactivity of the CpG due to a modification in backbone. Therefore, it is unclear whether this method would be optimal to structural modification of CpG ODNs.
The CpG ODN shows species-specificity and there has been no report that the CpG ODN shows a high immunoactivity in humans comparable to that it has shown in experimental animals. Further, since the action mechanism of CpG ODN is still unknown, it is hard to develop potent immunotherapeutic CpG ODN to target human immune cells. Consequently, there is very rare CpG ODN for a clinical trial. These limitations necessitate the development of CpG ODN either with superior immune-stimulating activity or lower side effects comparing to the existent CpG ODN.
The present inventors devised the simple method to solve the above problems. That is, a modified CpG ODN was prepared by coupling a consecutive sequence of deoxyribothymine (dT) to the 3′-terminus of CpG ODN, thus improving immunoactivity of splenocytes, macrophages and peripheral mononuclear cells, and therefore, can be effectively used as a vaccine adjuvant for preventing and treating hepatitis B or an anti-cancer agent.